This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Human parainfluenza virus 3 (HPIV3) is an enveloped virus containing a negative-sense single-stranded RNA genome of 15462 nucleotides long (GenBank Accession No. NC_001796), encoding a total of 8 proteins. The basic unit for genome transcription and replication is composed of nucleocapsid protein (N, 515 residues), phosphoprotein (P, 603 residues) and large protein (L, 2233 residues) together with the RNA genome. The N protein associates with genomic or anti-genomic RNA to form a stable RNase-resistant helical nucleocapsid. The P protein contains an oligomerization domain and is heavily phosphorylated. The L protein is a large multifunctional protein responsible for the catalytic activities of RNA transcription/replication. The P protein plays a key role in assembly of the RNA transcription/replication machinery. In addition to be able to self-assemble into homo-oligomers, the P protein can also associate with N and L, thereby linking L-P complex to the nucleocapsid to complete the assembly of the machinery. We are currently in the process of establishing a reverse genetics system for HPIV3. Once such a system is available, we will be able to tag each individual viral protein and purify them individually or in complex. Purification of nucleocapsid from HPIV3-infected cells will also be carried out. We have already established yeast and insect cell expression systems in the lab, and will produce helical nucleocapsids or ring-like nucleocapsids respectively. All the proposed complexes for single-particle cryo-EM analysis have molecular weights close or over 200 kD. We are confident that high quality purified samples will be obtained through affinity and size-exclusion chromatography. Detergent and high-salt concentration are not likely to be contained in the final buffer.